We will evaluate the potential of photogenerated reagents designed for investigations of transient phenomena (T1/2 miliseconds-minutes) associated with biological membranes. Four classes of reagents for elucidating the topography of membrane proteins will be studied: hydrophilic reagents, hydrophobic reagents, reagents that label at the membrane-buffer interphase and hydrophobic crosslinking reagents. On photolysis, the molecules commonly used as photoaffinity labels (e.g. aryl azides) give rise to both short-lived intermediates and undesirable long-lived intermediates. By combining the techniques of rapid quenching and flash photolysis, we will identify new reagents for which the contribution to labeling by long-lived intermediates is negligible and find conditions for the remaining reagents under which they can be efficiently quenched with chemical scavengers. Our ultimate goal is to label proteins in transient membrane states (such as the open form of an ion channel) by flash photolysis in a stopped-flow apparatus. Time-resolved, structural and topographic information will be obtained by mapping the labeling sites within the polypeptide chains of the membrane proteins.